Colon cancer currently accounts for 11% of all deaths due to malignancy annually in the United States. With an incidence of 62 per 100,000 and a prevalence of 300 per 100,000, the disease is currently the third leading cause of death in men and the fourth leading cause of death in women. Colon cancer has a particularly poor five-year survival rate of less than 50%, due to the late stage at which diagnosis is generally made. The currently favored treatment, surgery combined with chemotherapy, has failed to increase this survival rate. What is needed is a safe and effective preventive therapy which could be initiated early in patient populations known to be at an increased risk of developing colon cancer.
Eicosanoids and Differentiated Functions of Gastrointestinal Cells. Eicosanoids are significant regulators of gastrointestinal epithelial cell growth, differentiation and function. Eicosanoid products of the prostaglandin series are known to induce mucus secretion (Beckel and Kauffman (1981) Gastroenterology 80:770-776) and the secretion of electrolytes and fluid (Miller (1983) Am. J. Physiol. 245:G601-G623). They also induce active transport (Bukhave and Rask-Madsen (1980) Gastroenterology 78:32-37) and increase the replicative capacity of the epithelium (Konturek et al. (1981) Gastroenterology 80:1196-1201). These responses result in the maintenance of a differentiated, protective barrier system of tightly joined epithelial cells whose apical surface are covered by a dense glyco-conjugate chemical buffer. In the stomach and upper duodenum such a barrier protects against the acidic and proteolytic environment elaborated for digestion, while in the colon it protects against the invasion of bacteria and toxins. It is therefore not surprising that exogenous, synthetic prostaglandins are actively cytoprotective (Whittle and Vane (1987) In: Johnson (ed.) PHYSIOLOGY OF THE GASTROINTESTINAL TRACT, VOl. 1, 2nd ed., New York: Raven Press, pp. 143-180) and have found therapeutic utility as secondary anti-ulcer treatments. The gastrointestinal ("GI") system has therefore evolved to actively produce and rely on a specific differentiated complement of eicosanoid products present in the local environment. As all eicosanoids are derived from the common precursor arachidonic acid, which is itself liberated from membrane phospholipids, GI mucosal cells have a relatively high basal level of arachidonate turnover initiated by the enzyme phospholipase A.sub.2 (PLA.sub.2).
The GI system is also a primary defense mechanism against environmental bacteria, antigens and toxins and must therefore also possess the ability to mount an aggressive and rapid inflammatory response. This response also relies upon eicosanoid products of both the prostaglandin (PG) series as well as the chemotactic leukotriene series (LTs) and results in the influx of blood-borne neutrophils, macrophages and immune cells in response to the activating agent. Each of these invading cells also brings with it a capacity to metabolize its own phospholipids as well as mucosal and luminal phospholipids by releasing inflammatory (secreted) PLA.sub.2 to amplify the release of arachidonic acid, which is then metabolized into both PGs and LTs.
Although the inflammatory cell infiltration confines and destroys the offending stimulus, the extent of damage due to inflammatory cell release products is significant. Neutrophils and macrophages release superoxide (O.sub.2.sup.-) (Kitahora et al. (1988) Dig. Dis. Sci. 33:951-955) as well as hydrogen peroxide (H.sub.2 O.sub.2) (Tauber and Babior (1985) Free Radic. Biol. Med. 1:265-307), and proteases (Ohlsson et al. (1977) Hoppe Seylers Z. Physiol. Chem 358:361-366). Where the resultant infiltration is extensive, significant denudation of the epithelial layer occurs with a subsequent compromise of the barrier function. Resolution of the inflammatory response is then required to regain final optimal epithelial barrier coverage.
Chronic GI Inflammation and GI Cancer Induction. It is now well documented that chronic gastrointestinal inflammatory diseases such as ulcerative colitis (Lennard-Jones et al. (1977) Gastroenterology 73:1280-1285), Crohn's disease (Farmer et al. (1971) Cancer 28:289-295), and chronic atrophic gastritis (Sipponen et al. (1983) Cancer 52:1062-1067) are associated with an increased risk of subsequent gastrointestinal cancer. Although the mechanisms are not yet proven, three important intersecting pathways which could lead to subsequent transformation, heightened proliferation and malignant invasion occur during multiple acute inflammatory episodes. As discussed below these are: (1) increased colonic free radical and carcinogen load, (2) altered regulation of trophic eicosanoids, and (3) induction of gene products which mediate cellular invasion.
Altered carcinogen load due to inflammatory episodes. The colon may be uniquely exposed to a fairly high basal load of genotoxic carcinogens and tumor promoters resulting from the metabolism of dietary compounds and endogenous secretions such as bile acids by colonic bacteria. A relationship between fecal carcinogens and colon cancer induction is supported by findings of increased mutagens in the stools of high-risk individuals versus those of low-risk populations (Reddy et al. (1980) Mut. Res. 72:511-515). This correlation is also consistent with repeated findings showing a negative association between dietary fiber intake and incidence of colon cancer (Armstrong and Doll (1975) Int. J. Cancer 15:617-623). The protective effect of fiber is postulated to occur through an increase in stool volume which results in a dilution of stool carcinogens and decreased transmit time, leading to more rapid carcinogen elimination. These results raise the possibility that if a reduction in concentration of the stool carcinogen load can lead to a decreased cancer risk, then an increase in the carcinogen load may lead to an increased risk. One such increase in colonic carcinogens could be derived from successive inflammatory events.
A particularly relevant example is the inflammatory neutrophil production of carcinogenic nitrosamines via the L-arginine-dependent formation of nitrogen oxides as nitric oxide (Grisham (1993) Gastroenterology 104:A243). Other oxidative products released by inflammatory cells include superoxide as well as hydrogen peroxide, which in the presence of certain transition metals such as iron (Fe) can generate the highly reactive and cytotoxic hydroxyl radical (OH:) (Grisham (1990) Biochem Pharmacol 39:2060-2063). In addition to the increased carcinogen and free radical load elaborated by the influx of inflammatory cells, the arachidonic acid cascade is also known to be capable of producing mutagenic metabolites. A metabolite of prostaglandin H2 (PGH2), malondialdehyde (MDA), is a direct acting mutagen in vitro (Mukai and Goldstein (1976) Science 191:868-869) and a carcinogen in animals (Basu and Marnett (1983) Carcinogenesis 4:331-333) and can be enzymatically produced by thromboxane synthetase in high yields in cells with an active cyclooxygenase pathway. MDA has been shown to produce frameshift mutations similar to those associated with the human colon p53 gene (Marnett, et al. (1985) Mutat. Res. 129:36-46). PGH synthase itself is a potent peroxidase and has been shown to catalyze the activation of a wide range of polycyclic hydrocarbons to mutagens (Marnett (1981) Life Sci. 29:531-546).
These findings suggest that chronic and aberrant (inflammatory cell driven) activation of the arachidonic acid cascade in the gastrointestinal tract is one pathway which can lead to an increased carcinogen load with the potential induction of DNA mutations during times of maximum DNA synthesis. Increased cell proliferation, which follows epithelial denudation induced by invading inflammatory cells, could lead to an increased number of cells susceptible to the action of such carcinogens. Alternatively, increased proliferation could serve to amplify mutations (through clonal expansion) induced previously by carcinogens.
Altered eicosanoid regulation can drive proliferation due to inflammatory episodes. An additional mechanism linking GI inflammation and the progression of GI cancers could be a disruption in normal eicosanoid regulation. As discussed above, the normal differentiated functions of the GI mucosal epithelium are intimately linked to the diverse range of biological activities affected by endogenous eicosanoids. Since these agents act locally and generally have short half-lives due to active metabolic inactivation, periods of acute inflammatory events will dramatically alter the normal regulation of eicosanoid homeostasis.
Upon neutrophil and macrophage invasion into an inflammatory site these normal dynamics are dramatically altered. First, inflammatory cells bring with them a wide range of additional agonists such as cytokines, proteases and growth factors (Adams and Hamilton (1984) Ann. Rev. Immunol.2:283-318, Ohlsson et al. (1977) Physiol. Chem. 358:361-366, Nathan and Cohn (1980) In: Kelly et al. (eds.), Textbook of Rheumatology, New York: W. B. Saunders, pp. 186-215), which by themselves chronically activate cellular PLA.sub.2 (cPLA2) to release arachidonic acid . Second, inflammatory cells are a rich source of additional forms of PLA.sub.2 known as secretory or sPLA.sub.2 (Seilhamer et al. (1989) J. Biol. Chem. 264:5335-5338). whose activities in GI inflammatory disease has recently been documented (Minami et al. (1992) Gut 33:914-921).
Unlike cPLA.sub.2, sPLA.sub.2 is released from inflammatory cells (Wright et al. (1990) J. Biol. Chem. 265:6675-6681), platelets (Hayakawa et al. (1988) J. Biochem. 104:767-772), chondrocytes (Lyons-Goirdano et al. (1989) Biochem. Biopys. Res. Commun. 64:488-495) and smooth muscle cells (Nakano et al. (1990) FEBS Lett. 261:171-174) by cytokines (Pfeilscifter et al. (1989) Biophys. Res. Commun. 159:385-394), and, in particular, by endotoxin (Oka and Arita (1991) J. Biol. Chem. 266:9956-9960). In addition, because the extracellular milieu contains maximal calcium concentrations, sPLA.sub.2 is unregulated once released. Upon release it therefore actively hydrolyses arachidonic acid and other fatty acids from the sn-2 position of phospholipids found in cellular and bacterial membranes as well as those from dietary and lipoprotein sources. The lysophospholipid resulting from removal of the sn-2 fatty acid from many such phospholipids is potently lysogenic for surrounding cells (Okada and Cyong (1975) Jpn. J. Exp. Med. 45:533-534). Thus, this reaction can also lead to epithelial cell lysis and denudation in the unfiltered area, ultimately requiring enhanced proliferation to maintain the barrier function.
Inflammatory response and activation of genes directing cellular invasion. The inflammatory response not only disrupts normal eicosanoid regulation but also leads to the activation of gene products required for cellular invasion. One such product, the urokinase plasminogen activator receptor (uPAR), is normally expressed by intestinal epithelial cells. Its anchorage to the cell surface may be an important determinant of normal crypt cell migration and desquamation via cell surface proteolysis (Kristensen et al. (1991) J. Cell. Biol. 115:1763-1771). In inflammatory cells, uPAR gene expression is induced by activators of protein kinase C, by tumor promoters such as the phorbol ester TPA (Lund et al. (1991) J. Biol. Chem. 266:5177-5181), and by various cytokines (Lund et al. (1991) EMBO J. 10:3399-3401) which induce an invasive phenotype required for tissue infiltration (Stoppelli et al. (1985) Natl. Acad. Sci. USA 82:4939-4943). It is therefore not surprising that high levels of uPAR expression have also been detected in several tumor cell lines with metastatic potential, including colon cancer-derived cells (Pyke et al. (1991) Am. J. Pathol. 138:1059-1067). Of particular interest are co-culture experiments showing that invasive potential was more highly correlated with expression of uPAR than its ligand plasminogen activator (Ossowski et al. (1991) J. Cell. Biol. 15:1107-1112). Thus, multiple cycles of inflammatory responses could also contribute to uPAR overexpression in colonic mucosal cells, resulting in the acquisition of an invasive phenotype in a previously benign tumor.
GI mucosal cells may therefore be uniquely sensitive to chronic inflammatory episodes because of three intersecting pathways: (1) the epithelial cells are positioned in an environment with a high carcinogen load which can further increase during inflammatory episodes; (2) the products of both the endogenous and infiltrated eicosanoid cascades are trophic agents; and (3) their own differentiated response to inflammatory agents includes expression of gene products required for acquisition of an invasive phenotype. Together these three paths could lead to a transformation event and resultant tumor induction, progression and invasion. Therefore, agents which block certain arms of the eicosanoid cascade are useful in colon cancer chemoprevention.
NSAIDs and Colon Cancer Chemoprevention. There is evidence that several non-steroid antiinflammatory drugs (NSAIDs) are effective in reducing the number of tumorbearing animals and tumor incidence per animal in rat models of colon carcinogenesis. (Narisawa et al. (1981) Cancer Res. 41:1954-1957), (Pollard et al. (1983) Cancer Lett. 21:57-61) (Moorghen et al. (1988) J. Pathol. 156:341-347) (Reddy et al. (1993) Gastroenterology 104:A443) where up to a 70% reduction in tumor incidence has been noted at doses of 80% of the maximum tolerated dose. In a study of dimethylhydrazine-induced colon carcinogenesis, sulindac was observed to reduce tumor incidence only when present during carcinogen administration, but not if given 17 weeks following the carcinogen (Moorghen et al. (1988) J. Pathol. 156:341-347).
In several retrospective studies, aspirin intake has been assessed as a chemopreventive therapy in ensuing colon cancer incidence. The results of these studies have ranged from a halving (Kune et al. (1988) Cancer Res. 48:4399-4404) of the risk of colon cancer development to a 50% increased risk (Paganini-Hill et al. (1991) J. Natl. Cancer Inst. 83:1182-1183). It should be noted, however, that any human study employing aspirin and other NSAIDs, especially retrospective studies, could be flawed by the induced frequency of GI bleeding which may allow for earlier detection of the polyp or tumor in the NSAID group through occult blood screening and sigmoidoscopy.
Although the retrospective aspirin studies have provided equivocal results, the initial results for certain NSAIDs found in animal studies, summarized above, have been replicated in prospective human trials. The NSAID, sulindac, has been shown in a randomized, placebo-controlled, double-blind crossover study to cause regression of polyps in nine familial polyposis patients in less than 4 months (Labayle et al. (1991) Gastroenterology 101:635-639). In addition, polyp growth resumed following sulindac withdrawal. This finding is significant because a similar study using indomethacin found no influence on polyp regression (Klein et al. (1987) Cancer 60:2863-1868). Although both of these NSAIDs derive their anti-inflammatory activity from inhibition of cyclooxygenase, sulindac is a prodrug which is converted to its active metabolite, sulindac sulfide, by the colonic bacteria (Shen and Winter (1975) Adv. Drug. Res. 12:89-245). In contrast, indomethacin is ingested in its active form, where it is absorbed primarily from the upper GI tract for systemic delivery (Hucker et al. (1966) J. Pharmacol. Exp. Ther. 153:237-249). It is therefore likely that the concentrations of active metabolite delivered by sulindac to the colon are significantly higher than from indomethacin. This result suggests that a significant portion of the observed NSAID chemopreventive effect is derived from local action of the drug at the mucosal-lumen interface.
Although this evidence of NSAID-induced inhibition of colonic tumor incidence in animal models suggests a mechanism of inhibition via the tumor cell cyclooxygenase, confirmation of such a mechanism is lacking. Tumor tissue excised from NSAID-treated animals has been shown to secrete dramatically reduced levels of PGE.sub.2, which is consistent with this hypothesis (Reddy et al. (1992) Carcinogenesis 13:1019-1023). However, most colonic tumors are heterogeneous with respect to their resident cell types, and several reports have documented that epithelial cell lines generated from colorectal adenocarcinomas are not high producers of PGE.sub.2 or other prostanoids (Hubbard et al. (1988) Cancer Res. 48:4770-4775). A report on the eicosanoid production of cells fractionated from human colon tissue, however, showed that tumor epithelial cells produced PGE.sub.2 levels similar to uninvolved tissue, while tumor-derived mononuclear cells showed significantly greater eicosanoid synthesis than their normal counterparts (Maxwell et al. (1990) Digestion 47:160-166). Therefore, target cells sensitive to NSAID inhibition may not be the tumor epithelial cells, but could be some other resident producing high PG levels to which the epithelial cells are responsive.
Profile of the Preferred Chemopreventive Therapy All NSAIDs carry significant side effect profiles. NSAIDs are not tissue specific in their inhibition of cyclooxygenase products, and both renal and gastrointestinal systems are particularly sensitive. NSAIDs reduce renal perfusion resulting in nephrotoxicity (Clive and Stoff (1984) N. Engl. J. Med. 310:563-572), and because prostaglandins are necessary for the normal differentiated functions of the GI epithelium, NSAID induced gastric ulceration is a significant contributor to morbidity and mortality associated with this class of drugs (Langman (1989) Gastroenterology 96:640-646, Bjarnason et al. (1992) Gastroenterology 104:1832-1847). In the lower bowel, chronic NSAID therapy has been reported to result in colitis ranging from proctitis to pancolitis (Tanner and Raghunat (1988) Digestion 41:116-120). In one retrospective study, it was estimated that NSAID ingestion was found in 25% of patients presenting with large and small bowel perforation and bleeding (Langman et al. (1985) Br. Med. J. 290:347-349). Finally, in patients with existing chronic inflammatory bowel diseases, who are already at high risk of developing colorectal cancer, non-5-ASA-containing NSAIDs as well as aspirin therapy is contraindicated due to exacerbation of the existing disease condition (Rampton and Sladen (1981) Prostaglandins 21:417-425). The ideal drug for colon cancer chemoprevention is, therefore, one with the following profile:
1) Colon specific--It should be a prodrug with no activity elaborated in the upper GI tract, but converted to active form upon reaching the colon (sulindac-like); PA1 2) Limited absorption--Absorption of the parent and metabolites should be minimal, especially once converted to its active form; PA1 3) Lack of systemic activity--Once absorbed, metabolic inactivation should convert the drug to an inactive form, thereby limiting systemic effects on the renal and GI systems; PA1 4) Antioxidant properties--Colon-specific antioxidant activity would further serve to lower the carcinogen burden; and PA1 5) NSAID-like anti-inflammatory mechanism--The active metabolite should inhibit inflammation-induced eicosanoid pathways, however, eicosanoid inhibition without compromise to basal maintenance pathways would be preferred.
To summarize, certain NSAIDs have been shown to inhibit colon tumor incidence in carcinogen-induced animal models and inhibit polyp growth in humans. Although the mechanism for tumor inhibition by NSAIDs is unproven, these drugs may modulate gastrointestinal eicosanoid production and metabolism. Unfortunately, NSAIDs also have a disturbing and serious gastrointestinal side effect profile which may preclude their chronic use. Therefore, it would be highly desirable to identify NSAIDs which are effective as chemopreventive agents but which lack these side effects.
Chan, U.S. Pat. No. 4,412,992, describes the preparation of 2-hydroxy-5-phenylazobenzoic acid derivatives and their use in the treatment of ulcerative colitis.